Human rnase iii and compositions and uses thereof

ABSTRACT

The present invention provides polynucleotides encoding human RNase III and polypeptides encoded thereby. Methods of using said polynucleotides and polypeptides are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.11/001,993 filed Dec. 2, 2004, which is a divisional of U.S. applicationSer. No. 10/079,185 filed Feb. 20, 2002, which is a continuation-in-partof U.S. application Ser. No. 09/900,425 filed Jul. 6, 2001, U.S. Pat.No. 6,737,512. All of the above are assigned to the assignee of thepresent invention and are incorporated by reference herein in theirentirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledISIS5030USC1SEQ.txt, created on Dec. 22, 2008 which is 36 Kb in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a human RNase III, the gene for whichhas now been cloned and characterized, and compositions and usesthereof. Antisense inhibitors of human RNase III are also described.

BACKGROUND OF THE INVENTION

Ribonuclease III (RNase III) is an endoribonuclease that cleaves doublestranded RNA. The enzyme is expressed in many organisms and is highlyconserved. I. S. Mian, Nucleic Acids Res., 1997, 25, 3187-95. All RNaseIII species cloned to date contain an RNase III signature sequence andvary in size from 25 to 50 kDa. Multiple functions have been ascribed toRNase III. In both E. coli and S. cerevisiae, RNase III has beenreported to be involved in the processing of pre-ribosomal RNA(pre-rRNA). Elela et al., Cell, 1996, 85, 115-24. RNase III has alsobeen reported to be involved in the processing of small molecular weightnuclear RNAs (snRNAs) and small molecular weight nucleolar RNAs(snoRNAs) in S. cerevisiae. Chanfreau et al., Genes Dev. 1996, 11,2741-51; Qu et al., Mol. Cell. Biol. 1996, 19, 1144-58. In E. coli,RNase III has also been reported to be involved in the degradation ofsome mRNA species. D. Court, in Control of messenger RVA stability,1993, Academic Press, Inc, pp. 71-116.

A human double strand RNase (dsRNase) activity has been described. Wu etal., J. Biol. Chem., 1998, 273, 2532-2542; Crooke, U.S. Pat. No.5,898,031; U.S. Pat. No. 6,017,094. By the rational design and testingof chemically modified antisense oligonucleotides that containedoligoribonucleotide stretches of varying length, a dsRNase activity wasdemonstrated in human T24 bladder carcinoma cells which produced5′-phosphate and 3′-hydroxyl termini upon cleavage of the complementarycellular RNA target. This pattern of cleavage products is a feature ofE. coli RNase III. The cleavage activity in human cells required theformation of a dsRNA region in the oligonucleotide. This human dsRNaseactivity is believed to be useful as an alternative terminatingmechanism to RNase H for antisense therapeutics. Because it relies on“RNA-like” oligonucleotides, which generally have higher potency thanthe “DNA-like” oligonucleotides required for RNase H activity, it mayprove an attractive alternative to RNase H-based antisense approaches.

RNA interference (RNAi) is a form of sequence-specific,post-transcriptional gene silencing in animals and plants, elicited bydouble-stranded RNA (dsRNA) that is homologous in sequence to thesilenced gene. Elbashir et al., Nature, 2001, 411, 494-498. dsRNAtriggers the specific degradation of homologous RNAs, only within theregion of homology. The dsRNA is processed to 21- to 23-nucleotidefragments, sometimes called short interfering RNAs (siRNAs) which arebelieved to be the guide fragments for sequence-specific mRNAdegradation. The processing of longer dsRNA to these short siRNAfragments is believed to be accomplished by RNase III. Elbashir et al.,ibid., Elbashir et al., Genes and Devel., 2001, 15, 188-200. Thus it isbelieved that the human RNase III of the present invention may be usefulin further understanding and exploiting the gene silencing mechanism,particularly in human cells.

Despite the substantial information about members of the RNase IIIfamily and the cloning of genes encoding proteins with RNase IIIactivity from a number of lower organisms (E. coli, yeast and others),no human RNase III has previously been cloned. This has hampered effortsto understand the structure of the enzyme(s), its distribution and thefunctions it may serve. The present application describes the cloningand characterization of a cDNA that expresses a human RNase III. Cloningand sequencing of the cDNA encoding human RNase III allowedcharacterization of this nucleic acid as well as of the location andfunction of the RNase III protein itself.

SUMMARY OF THE INVENTION

The present invention provides a polynucleotide sequence (set forthherein as SEQ ID NO: 1) which has been identified as encoding humanRNase III by the homology of the calculated expressed polypeptide(provided herein as SEQ ID NO: 2) with known amino acid sequences ofyeast and worm RNase III as well as by functional analysis.

The present invention provides polynucleotides that encode human RNaseIII, the human RNase III polypeptide, vectors comprising nucleic acidsencoding human RNase III, host cells containing such vectors, antibodiestargeted to human RNase III, nucleic acid probes capable of hybridizingto a nucleic acid encoding a human RNase III polypeptide, and antisenseinhibitors of RNase III expression. Methods of inhibiting RNase IIIexpression or activity are also provided, as are pharmaceuticalcompositions which include a human RNase III polypeptide, an antisenseinhibitor of RNase III expression, or a vector containing a nucleic acidencoding human RNase III.

Methods for identifying agents which modulate activity and/or levels ofhuman RNase III are also provided. Methods of promoting inhibition ofexpression of a selected protein via antisense, methods of screeningoligonucleotides to identify active antisense oligonucleotides against aparticular target, methods of prognosticating efficacy of antisensetherapy, methods of promoting RNA interference (RNAi) or other forms ofgene silencing in a cell and methods of eliciting cleavage ormodification of a selected cellular RNA target are also provided. All ofthese methods exploit the RNA-binding and cleaving activity of RNase IIIpolypeptides. In preferred embodiments the polynucleotides used in thesemethods are RNA-like oligonucleotides. Also provided are methods ofidentifying agents which increase or decrease activity or levels ofhuman RNase III.

The compositions and methods of the present invention are useful forresearch, biological and clinical purposes. For example, the methods,polynucleotides and antisense oligonucleotides are useful in definingthe roles of RNase III and the interaction of human RNase III andcellular RNA (including pre-mRNA or pre-rRNA).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of human RNase III (SEQ ID NO: 2)and a comparison of the sequence of the RNase III domain of the humanRNase III to RNase III domains of C. elegans (Worm; SEQ ID NO: 3), S.pombe (PAC; SEQ ID NO: 4) and S. cerevisiae (RNT; SEQ ID NO: 5) and E.coli (RNC; SEQ ID NO: 6). Bold letters: identical amino acids of humanRNase III to other species. @@@: putative catalytic center. HHH: alphahelix; BBB: beta sheet (dsRNA binding region at C-terminus). Amino acididentity of human RNase III to Worm (41%), PAC (17%), RNT (15%) and RNC(16%). *: Potential phosphorylation sites analyzed using OMIGA (OxfordMolecular Ltd.).

DETAILED DESCRIPTION OF THE INVENTION

A cDNA encoding human RNase III has now been cloned and characterized.The cloned sequence is provided herein as SEQ ID NO: 1. This cDNAencodes a protein of 160 kDa which is ubiquitously expressed in humancell and tissue types, and is involved in processing of preribosomal RNA(pre-rRNA).

Thus, in accordance with one aspect of the present invention, there areprovided isolated polynucleotides which encode human RNase IIIpolypeptides. By “polynucleotides” it is meant to include any form ofRNA or DNA such as mRNA, pre-mRNA or cDNA or genomic DNA, respectively,obtained by cloning or produced synthetically by well known chemicaltechniques. The term “polynucleotide” is also meant to includeoligonucleotides, e.g. synthetic antisense oligonucleotides. DNA or RNApolynucleotides may be double- or single-stranded. Single-stranded DNAor RNA polynucleotides may comprise the coding or sense strand or thenon-coding or antisense strand.

Methods of isolating a polynucleotide of the present invention viacloning techniques are well known. For example, to obtain thepolynucleotide sequence of SEQ ID NO: 1, a similarity search of theyeast RNTI gene (RNase III, Genbank accession no. AABO4172; SEQ ID NO:5) and the Caenorhabditis elegans RNase III gene (Genbank accession no.001326; SEQ ID NO: 3) with the XREF database (National Center forBiotechnology Information, NIH, Rockville Md.) was performed. A 393 basepair (bp) human EST clone (GenBank AA083888) was identified.

Using primers based on this EST sequence, a clone (U4) corresponding tothe COOH-terminal portion of the protein (nucleotides 3569-4764 of fulllength cDNA) was cloned by 3′ RACE. Eight positive clones were isolatedby screening a liver cDNA library with this clone. With primers based onone of these clones, 5′ RACE was performed to clone a cDNA ofapproximately 1 kb, which corresponds to the middle part of the fulllength cDNA. In the same way, a cDNA of the NH₂-terminal portion wascloned. Primers based on the NH₂-terminal-most clone were used toperform additional 5′-RACE to obtain the NH₂-terminal portion of thecDNA. The overlapping clones were sequenced and assembled to a fulllength human RNase III cDNA with a total of 4764 nucleotides. This humanRNase III polynucleotide sequence is provided herein as SEQ ID NO: 1 andhas been deposited as GenBank accession no. AF189011. The cDNA containeda coding sequence of 4125 nucleotides (from 246-4370 of SEQ ID NO:1)that was calculated to encode a 1374 amino acid protein. Thispolypeptide sequence is provided herein as SEQ ID NO: 2, shown inFIG. 1. The calculated molecular weight of the protein is 160 kDa basedon the prediction of the first translated methionine as the translationinitiation site. Northern hybridization analyses demonstrated that thehuman RNase III mRNA was approximately 5 kb in size. It was found to beubiquitously expressed in human tissues and cell lines. Compared to C.elegans, yeast and bacterial RNase III, human RNase III is substantiallylarger and contains multiple domains. The RNase III domain (amino acids949-1374) is located at the carboxy terminus of the protein and ishomologous to C. elegans, yeast and bacterial RNase III. The human RNasealso contains proline rich (amino acids 1-220) and serine-arginine rich(amino acids 221-470) domains near the amino terminus. The SR and RNaseIII domains are separated by 478 amino acids.

The RNase III domain of human RNase III is conserved with other speciesand is most homologous with C. elegans RNase III (41% identity). Boththe human RNase III domain and C. elegans RNase III contain two RNaseIII signature sequences (HNERLEFLGDS; SEQ ID NO 7). Sequence identitywas also compared with the yeasts S. pombe (PAC gene)(17% homology) andS. cerevisiae (RNT gene) (15% homology) and with E. coli RNase III (RNCgene) (16% homology). Human RNase III also contains multiplephosphorylation sites. The SR domain is usually present in SR or SRrelated proteins that play crucial roles in mRNA splicing. The fusion ofSR and RNase III domains into a single protein suggests that human RNaseIII may be involved in a number of RNA metabolic events. The presence ofmultiple potential phosphorylation sites suggests that the enzyme isregulated by phosphorylation.

As used herein, the phrase “homologous nucleotide sequence,” or“homologous amino acid sequence,” or variations thereof, refers tosequences characterized by a homology, at the nucleotide level or aminoacid level, of at least the specified percentage. Homologous nucleotidesequences include those sequences coding for isoforms of proteins. Suchisoforms can be expressed in different tissues of the same organism as aresult of, for example, alternative splicing of RNA. Alternatively,isoforms can be encoded by different genes. Homologous nucleotidesequences include nucleotide sequences encoding for a protein of aspecies other than humans, including, but not limited to, mammals.Homologous nucleotide sequences also include, but are not limited to,naturally occurring allelic variations and mutations of the nucleotidesequences set forth herein. A homologous nucleotide sequence does not,however, include the nucleotide sequence encoding other known RNaseIIIs. Homologous amino acid sequences include those amino acid sequenceswhich contain conservative amino acid substitutions and whichpolypeptides have the same binding and/or activity. A homologous aminoacid sequence does not, however, include the amino acid sequenceencoding other known RNase IIIs. Percent homology can be determined by,for example, the Gap program (Wisconsin Sequence Analysis Package,Version 8 for Unix, Genetics Computer Group, University Research Park,Madison Wis.), using the default settings, which uses the algorithm ofSmith and Waterman (Adv. Appl. Math., 1981, 2, 482-489, which isincorporated herein by reference in its entirety).

In a preferred embodiment, the polynucleotide of the present inventioncomprises the nucleic acid sequence of SEQ ID NO: 1. However, as will beobvious to those of skill in the art upon this disclosure, due to thedegeneracy of the genetic code, polynucleotides of the present inventionmay comprise other nucleic acid sequences encoding the polypeptide ofSEQ ID NO: 2 and derivatives, variants or active fragments thereof.

The invention further provides homologs of the human RNase III DNA. Suchhomologs, in general, share at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99% homology with the human RNaseIII DNA of the invention. Species homologs, sometimes referred to as“orthologs,” in general, share at least 50%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99% homology with the human RNaseIII DNA of the invention. Generally, percent sequence “homology” withrespect to polynucleotides of the invention can be calculated as thepercentage of nucleotide bases in the candidate sequence that areidentical to nucleotides in the human RNase III sequence set forth inthe appended Sequence Listing, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity.

Another aspect of the present invention relates to the polypeptidesencoded by the polynucleotides of the present invention. In a preferredembodiment, a polypeptide of the present invention comprises the deducedamino acid sequence of human RNase III provided in SEQ ID NO: 2.However, by “polypeptide” it is also meant to include fragments,derivatives and analogs of SEQ ID NO: 2 which retain essentially thesame biological activity and/or function as human RNase III.Alternatively, polypeptides of the present invention may retain theirability to bind to double stranded RNA even though they do not functionas active RNase III enzymes in other capacities. Thus an enzyme may“modify” its RNA substrate, e.g., bind and interfere with the functionof the RNA but not cleave it, or may bind and cleave. In someembodiments cleavage is a preferred form of modification. In anotherembodiment, polypeptides of the present invention may retain nucleaseactivity but without specificity for an RNA/RNA duplex. Polypeptides ofthe present invention include recombinant polypeptides, isolated naturalpolypeptides and synthetic polypeptides, and fragments thereof whichretain one or more of the activities described above.

The invention further provides homologs of the human RNase IIIpolypeptide. Such homologs, in general, share at least 50%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% homologywith the RNase III polypeptides of the invention. Generally, percentsequence “homology” with respect to polypeptides of the invention can becalculated as the percentage of amino acid residues in the candidatesequence that are identical to amino acid residues in the RNAse IIIsequences set forth in the appended Sequence Listing, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity.

In some embodiments the present invention provides recombinantpolypeptides that comprise RNase III domains from one or more organisms.Domains of RNase III that exhibit certain functions can be replaced withRNase III domains from other organisms that exhibit similar functions,while maintaining the overall function of the polypeptide. As anon-limiting example, a hybrid RNase III may comprise one or more E.coli RNase III domain, one or more C. elegans RNase III domain, and oneor more human RNase III domain. As a non-limiting example, such hybridRNase III polypeptides can be produced by first designing and producingrecombinant DNA molecules encoding such polypeptides. Such recombinantDNA molecules are produced, for example, by replacing DNA sequences thatencode individual domains in a polynucleotide encoding RNase III withDNA sequences from other organisms that encode RNase III domains thatexhibit similar functions. The recombinant DNA construct thus producedcan then be expressed and purified using means familiar to one ofordinary skill in the art.

To confirm the expression of the human RNase III protein, twoanti-peptide antibodies were produced. The “anti-III” peptide antibodywas derived from a peptide corresponding to amino acids 1356-1374 withinthe RNase III domain present in the C-terminal portion of the putativeprotein. The “anti-SR” peptide antibody was derived from a peptidecorresponding to amino acids 266-284 within the SR-domain of theputative protein. Using these antibodies, Western blot analyses wereperformed to determine the size and localization of human RNase III. Theanti-SR peptide antibody recognized a band in HeLa whole cell lysatewith a molecular weight of approximately 160 kDa which is near thecalculated protein size confirming that the full coding region isexpressed in HeLa cells. Similar experiments were performed usingdifferent human cell lines e.g. A549, T24 and HL60 with equivalentresults. To determine the localization of the protein, nuclear andnon-nuclear fractions from HeLa cells and other human cell lines wereprepared and equal amounts of proteins were analyzed by Western blots.RNase III was present primarily in the nuclear fractions. Non-nuclearfractions contained only trace amounts of protein, possibly due to thecontamination during sample preparation. The anti-III peptide antibodygave results equivalent to those obtained with the anti-SR peptideantibody. To better understand the localization of human RNase III, theprotein was identified in cells by indirect immunofluorescencemicroscopy. The nuclei of HeLa cells were stained by both anti-SR andanti-III antibodies, confirming that human RNase III is present in thenucleus. RNase III is localized extensively in nucleus and occasionallyobserved in nucleoli. This localization suggests possible involvement inboth pre-mRNA and pre-rRNA processing. In E. coli, RNase III isassociated with ribosomes in the cytoplasm. Robertson et al., J. Biol.Chem., 1968, 243, 82-91. Eukaryotic RNase III has not previously beenshown to be localized in the nucleus.

The localization of human RNase III to nucleoli was found to be cellcycle regulated. Double thymidine treatment was used to synchronize HeLacells to early-S phase. Two to four hours after releasing the thymidineblock, HeLa cells entered S phase as determined by fluorescenceactivated cell sorting (FACS). Six to eight hours after release, HeLacells entered the G2/M phase. There were no significant changes in themRNA or protein levels of the RNase III during pre-S, S or G2/M phases.However, the subcellular localization of the protein changed during thecell cycle. When the cells were treated with thymidine and synchronizedin early S phase, RNase III protein was present only in the nucleus andnot the nucleoli, as determined by immunofluorescent labeling. Afterreleasing from thymidine block, RNase III was translocated to nucleoli,reaching a peak at 4 hours when cells were in S phase. At that time,RNase III was present both in the nucleoli and the nucleus. The proteinwas present in the nucleoli for approximately 8 hours, and thendisappeared from nucleoli as cells entered M phase. Localization ofRNase III in the nucleoli was confirmed by double staining with ananti-nucleolin monoclonal antibody (MBL, Watertown, Mass.).

In human cells, nucleoli undergo phases of condensation and dissociationas a function of the cell cycle. Nucleoli dissociate upon enteringprophase and disappear entirely during the late prophase and metaphaseperiods of mitosis, then begin to reappear during telophase and formdense organelles during the G1 phase. Human RNase III was onlytranslocated to and remained in the nucleoli during S phase suggestingthat RNase III may serve one or more specific functions in nucleoliduring S phase.

The present invention also provides antisense inhibitors of RNase IIIexpression, which may be used, for example, therapeutically,prophylactically or as research reagents. The modulation of function ofa target nucleic acid (in this case a nucleic acid encoding RNase III)by compounds which specifically hybridize to it is generally referred toas “antisense”. The functions of DNA to be interfered with includereplication and transcription. The functions of RNA to be interferedwith include all vital functions such as, for example, translocation ofthe RNA to the site of protein translation, translation of protein fromthe RNA, splicing of the RNA to yield one or more mRNA species, andcatalytic activity which may be engaged in or facilitated by the RNA.The overall effect of such interference with target nucleic acidfunction is modulation of the expression of the target. In the contextof the present invention, “modulation” means either an increase(stimulation) or a decrease (inhibition) in the expression of a gene. Inthe context of the present invention, inhibition is the preferred formof modulation of gene expression and mRNA is a preferred target. In someembodiments gene silencing is a preferred form of inhibition of geneexpression and refers to a decrease in gene expression mediated by adouble-stranded RNA polynucleotide, one strand of which is homologous tothe RNA to be silenced.

It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. The targeting process also includes determination of asite or sites within this gene for the antisense interaction to occursuch that the desired effect, e.g., detection or modulation ofexpression of the protein, will result. Within the context of thepresent invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of the target, regardless of the sequence(s) ofsuch codons.

It is also known in the art that a translation termination codon (or“stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA,5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAGand 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Other target regions include the 5′ untranslatedregion (5′UTR), known in the art to refer to the portion of an mRNA inthe 5′ direction from the translation initiation codon, and thusincluding nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. It has also been found thatintrons can also be effective, and therefore preferred, target regionsfor antisense compounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

Antisense and other compounds of the invention which hybridize to thetarget and inhibit expression of the target are identified throughexperimentation, and the sequences of these compounds are hereinbelowidentified as preferred embodiments of the invention. The target sitesto which these preferred sequences are complementary are hereinbelowreferred to as “active sites” and are therefore preferred sites fortargeting. Therefore another embodiment of the invention encompassescompounds, including primers, probes, siRNAs, other double stranded RNAsincluding RNAi or gene silencing agents, ribozymes, external guidesequence (EGS) oligonucleotides (oligozymes), and other short catalyticRNAs or catalytic oligonucleotides which hybridize to these activesites.

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat oligonucleotides can be useful therapeutic modalities that can beconfigured to be useful in treatment regimes for treatment of cells,tissues and animals, especially humans.

In the context of this invention, the term “polynucleotide”, whichincludes oligonucleotides, refers to an oligomer or polymer ofribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimeticsthereof. This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

In general, nucleic acids or polynucleotides (includingoligonucleotides) may be described as “DNA-like” (i.e., having 2′-deoxysugars and, generally, T rather than U bases) or “RNA-like” (i.e.,having 2′-hydroxyl or 2′-modified sugars and, generally U rather than Tbases). Nucleic acid helices can adopt more than one type of structure,most commonly the A- and B-forms. It is believed that, in general,oligonucleotides which have B-form-like structure are “DNA-like” andthose which have A-form-like structure are “RNA-like”.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. The antisense compounds in accordance with thisinvention preferably comprise from about 8 to about 50 nucleobases (i.e.from about 8 to about 50 linked nucleosides). Particularly preferredantisense compounds are antisense oligonucleotides, even more preferablythose comprising from about 12 to about 30 nucleobases. Antisensecompounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric structure can be further joined to form a circular structure,however, open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotideshaving inverted polarity comprise a single 3′ to 3′ linkage at the3′-most internucleotide linkage i.e. a single inverted nucleosideresidue which may be abasic (the nucleobase is missing or has a hydroxylgroup in place thereof). Various salts, mixed salts and free acid formsare also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697; and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

A further preferred modification includes Locked Nucleic Acids (LNAs) inwhich the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of thesugar ring thereby forming a bicyclic sugar moiety. The linkage ispreferably a methelyne (—CH₂—), group bridging the 2′ oxygen atom andthe 3′ or 4′ carbon atom wherein n is 1 or 2. LNAs and preparationthereof are described in WO 98/39352 and WO 99/14226.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one).

Modified nucleobases may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. The compounds of the invention caninclude conjugate groups covalently bound to functional groups such asprimary or secondary hydroxyl groups. Conjugate groups of the inventioninclude intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugates groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992 theentire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-5-tritylthiol(Manoharan et al., Ann. NY. Acad. Sci., 1992, 660, 306-309; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present inventionpreferably includes antisense compounds which are chimeric compounds.“Chimeric” antisense compounds or “chimeras,” in the context of thisinvention, are antisense compounds, particularly oligonucleotides, whichcontain two or more chemically distinct regions, each made up of atleast one monomer unit, i.e., a nucleotide in the case of anoligonucleotide compound. These oligonucleotides typically contain atleast one region wherein the oligonucleotide is modified so as to conferupon the oligonucleotide increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligonucleotide mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids.

By way of example, RNase H cleaves the RNA strand of an RNA:DNA duplex.Activation of RNase H, therefore, results in cleavage of the RNA target,thereby greatly enhancing the efficiency of oligonucleotide inhibitionof gene expression. Consequently, comparable results can often beobtained with shorter oligonucleotides when chimeric oligonucleotidesare used, compared to phosphorothioate deoxyoligonucleotides hybridizingto the same target region. Oligonucleotides, particularly chimericoligonucleotides, designed to elicit target cleavage by RNase H, thusare generally more potent than oligonucleotides of the same basesequence which are not so optimized. Cleavage of the RNA target can beroutinely detected by, for example, gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

Chimeric oligonucleotides may have one or more modifications of theinternucleoside (backbone) linkage, the sugar or the base. In apreferred embodiment, the oligonucleotide is a chimeric oligonucleotidehaving a modification at the 2′ position of at least one sugar moiety.Presently believed to be particularly preferred are chimericoligonucleotides which have approximately four or more deoxynucleotidesin a row, which provide an RNase H cleavage site, flanked on one or bothsides by a region of 2′-modified oligonucleotides.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

Antisense inhibition of human RNase III expression was used to furtherevaluate the role(s) of RNase III. To identify optimal sites in RNaseIII mRNA for antisense effects, 2′-O-methoxyethyl chimeric antisenseoligonucleotides targeted to 10 sites in the mRNA were designed andscreened for inhibition of RNase III. These are shown in Table 1. Thesechimeric or “gapped” oligonucleotides are designed to serve assubstrates for RNase H when bound to RNA resulting in degradation of thetarget RNA and oligonucleotides of this type have been shown to behighly specific when used under the described conditions.

TABLE 1 Antisense inhibition of human RNase III SEQ Target % ID ISIS #Sequence (5′-->3′) sites Inhib'n NO: 25690 ATCCCTTTCTTCCGCATGTG3051-3070 79 8 25691 GCCAAGGCGTGACATGATAT 3085-4004 96 9 25692CGGATCATTAAAGAGCAAGC 3442-3461 78 10 25693 TATTCACCAAAGAGCTTCGC3776-3795 49 11 25694 CAATCGTGGAAAGAAGCAGA 3973-3992 50 12 25695GCTCCCATTTCCGCTTGCTG 4197-4216 81 13 25696 ATGCTCTCTTTCCCACCTCA4308-4327 70 14 25697 AAATACTCCACACTTGCATG 4378-4397 79 15 25698TGCACATTCACCAAAGTCAA 4420-4439 44 16 25699 AGTCTAGGGTCACAATCTGG4688-4707 31 17 27110 TTCAGTTGTAGTGGTCCGAC 3-mismatch N/D 18 of 25691All oligonucleotides in Table 1 have phosphorothioate (P═S or PS)backbones and 2′-methoxyethoxy (2′MOE) “wings” flanking a 2′deoxy gap.2′MOE nucleotides are shown in bold. All cytosines are 5-methylcytosines (5meC). Target site refers to nucleotide numbers on the clonedRNase III cDNA (SEQ ID NO: 1) to which the oligonucleotide binds.Oligonucleotide concentration was 200 nM.

Table 1 shows that ISIS 25690, 25691, 25692, 25693, 25694, 25695, 25696and 25697 (SEQ ID NO: 8, 9, 10, 11, 12, 13, 14 and 15) inhibited humanRNase III expression by about 50% or more. These compounds are thereforepreferred. The most effective agent was ISIS 25691 (SEQ ID NO: 9),targeted to nucleotides 3085-4004 in the coding region of the mRNA. Thiscompound was selected for further studies.

Increasing concentrations of ISIS 25691 caused increasing loss of RNaseIII mRNA, with 300 nM resulting in loss of more than 90% of the RNaseIII mRNA. The mismatch control oligonucleotide, ISIS 27110 (SEQ ID NO:18), at 300 nM had no effect on the RNase III mRNA level. ISIS 25691 at300 nM suppressed RNase III mRNA levels in HeLa cells from 2 to 72 hoursafter a single treatment. After treatment with ISIS 25691 at 100, 150 or200 nM for 24 hours, RNase III protein was reduced to 67%, 44% or 19% ofcontrol respectively. The level of RNase III protein was slightlyreduced at 5 hours after treatment and reached a maximum reduction ofabout 70% at 18 hours.

Immunofluorescence staining showed that after treatment with ISIS 25691(150 nM, 24 hours), RNase III was dramatically reduced or absent in thenucleus and nucleoli. After treatment of HeLa cells with ISIS 25691 at300 nM for 18 hours, the morphology of HeLa cells changed from fusiformto oval. After 24 hours of treatment, approximately 5-10% of the cellsdetached from the plate and could be stained with trypan blue indicatingcell death. The cells that remained attached to the solid substrate grewmuch more slowly than untreated cells and appeared unable to entermitosis (data not shown). After 48 hours, 40-50% of the cells treatedwith 300 nM ISIS 25691 were dead. These results were highly reproducibleand indicate that RNase III is required for HeLa cell survival. Thecontrol oligonucleotide had no effect at any time or at anyconcentration on cell morphology, RNase III mRNA or protein levelsdemonstrating the antisense effect was highly specific.

One function that has been attributed to RNase III in lower species ispre-ribosomal RNA (pre-rRNA) processing. Human pre-rRNA processing isthought to involve cleavage of 45S pre-rRNA into 30S and 32S fragments.The 32S RNA product of the cleavage of 45S pre-rRNA contains 5.8S rRNA,ITS2 and 28S rRNA. Cleavage of the 32S RNA results in 12S pre-rRNA and28S rRNA products. The 12S pre-rRNA is further cleaved to 5.8S rRNA.Because ribosomes are made in the nucleolus, and the human RNase IIIprotein appeared to be translocated to and from the nucleolus during thecell cycle, its potential role(s) in human pre-rRNA processing wasevaluated. Two hybridization probes for human pre-rRNA were synthesized,5′ETS-1 (5′-CAA GGC ACG CCT CTC AGA TCG CTA GAG AAG GCT TTT CTC A-3′;SEQ ID NO: 19), designed to bind to the 5′ external transcribed spacer(5′ETS) of human pre-rRNA and 5.8S-1 (5′-CAT TAA TTC TCG CAG CTA GCG CTGCGT TCT TCA TCG ACG C-3′; SEQ ID NO: 20), designed to bind to 5.8S rRNA.When total cellular RNA (15 μg) from untreated HeLa cells wasfractionated by agarose gel electrophoresis, transferred to a nylonmembrane and probed with ³²P-5′ETS-1, a band corresponding to 45Spre-rRNA and a very faint band corresponding in mobility to 30S(5′ETS-18S-ITS1) pre-rRNA were observed. When ³²P-5.8S-1 was used, bandscorresponding to 45S, 32S (5.8S-ITS2-28S) and 12S (5.8S-ITS2) pre-rRNAand 5.8S rRNA were observed. At concentrations at which the antisenseoligonucleotide ISIS 25691 dramatically reduced the RNase III level, noeffect on the 45S pre-rRNA level was observed. In contrast, the 5.8S-1probe demonstrated that antisense inhibition of RNase III increased thelevels of 32S and 12S pre-rRNAs.

To provide further confirmation that human RNase III is involved inpreribosomal RNA processing, the effects of ten antisenseoligonucleotides on RNase III mRNA levels were compared to the effectsof these oligonucleotides on accumulation of the two pre-rRNA species(32S and 12S) that accumulated after treatment with the most potent ofthe antisense inhibitors, ISIS 25691. The potency of antisenseinhibitors designed to bind to different sites in RNase III mRNA varied.The correlation between the reduction of RNase III RNA levels and theaccumulation of both 32S and 12S pre-rRNAs was excellent, thusconfirming the conclusion derived from the Northern blot analysis.

Antisense inhibition of RNase III resulted in substantial accumulationof 12S pre-rRNA, less pronounced accumulation of 32S pre-rRNA and noaccumulation of 45S pre-rRNA. Thus this human RNase III appears to berequired for the processing of 12S pre-rRNA. It may also be involved inthe processing of 32S pre-rRNA. The principal site of cleavage inducedby human RNase III described here is in the 5.8S-ITS2 region ofpre-rRNA.

RNase III enzymes are double-strand RNA (dsRNA) endoribonucleases. Totest whether the human RNase III domain can specifically cleave dsRNA,the RNase III domain-coding region was subcloned into a glutathioneS-transferase (GST) expression vector. The GST-RNase III fusion proteinand GST alone were expressed, purified using glutathione agarose andanalyzed by coomassie blue staining of the SDS-PAGE and Western Blotanalysis with anti-human RNase III peptide antibody. These studiesshowed that the human RNase III domain was greater than 85% pure, thoughthere was evidence of slight degradation during expression andpurification. When incubated with labeled dsRNA and ssRNA, the GST-RNaseIII fusion protein preferentially digested the dsRNA without significantcleavage of ssRNA, while GST alone cleaved neither dsRNA nor ssDNAsubstrate. Thus, the cleavage observed was not due to contamination withssRNases or dsRNases from E. coli. Ribonucleases V₁ (dsRNase), and T₁and A (ssRNases) were used as controls to confirm that the cleavageobserved was dsRNA cleavage.

RNase III is a double-strand RNA endonuclease, specifically cleavingdouble-helical structures in cellular and viral RNAs. It is believedthat this cleavage can be exploited to promote cleavage of a cellularRNA target, by providing “RNA-like” antisense oligonucleotides whichhybridize to the cellular RNA target to form an RNA duplex, thuseliciting RNase III cleavage. Methods of promoting inhibition ofexpression by antisense oligonucleotides, and methods for screeningoligonucleotides are thus provided. In the context of this invention,“promoting antisense inhibition” or “promoting inhibition of expression”of a selected RNA target, or of its protein product, means inhibitingexpression of the target or enhancing the inhibition of expression ofthe target. In some embodiments of these methods, the RNase III ispresent in an enriched amount. In the context of this invention,“enriched” means an amount greater than would naturally be found. RNaseIII may be present in an enriched amount through, for example, additionof exogenous RNase III, through selection of cells which overexpressRNase III or through manipulation of cells to cause overexpression ofRNase III. The exogenously added RNase III may be added in the form of,for example, a cellular or tissue extract, a biochemically purified orpartially purified preparation of RNase III, or a cloned and expressedRNase III polypeptide.

The expression of large quantities of a cloned human RNase III of thepresent invention has been shown to be useful in characterizing theactivities of this enzyme. In addition, the polynucleotides andpolypeptides of the present invention provide a means for identifyingagents, such as the antisense compounds described herein, which modulatethe function of this enzyme in human cells and tissues. For example, ahost cell can be genetically engineered to incorporate polynucleotidesand express polypeptides of the present invention. Polynucleotides canbe introduced into a host cell using any number of well known techniquessuch as infection, transduction, transfection or transformation. Thepolynucleotide can be introduced alone or in conjunction with a secondpolynucleotide encoding a selectable marker. In a preferred embodiment,the host comprises a mammalian cell. Such host cells can then be usednot only for production of human RNase III, but also to identify agentswhich increase or decrease levels of expression or activity of humanRNase III in the cell. In these assays, the host cell would be exposedto an agent suspected of altering levels of expression or activity ofhuman RNase III in the cells. The level or activity of human RNase IIIin the cell would then be determined in the presence and absence of theagent. Assays to determine levels of protein in a cell are well known tothose of skill in the art and include, but are not limited to,radioimmunoassays, competitive binding assays, Western blot analysis andenzyme linked immunosorbent assays (ELISAs). Methods of determiningincreased activity of the enzyme, and in particular increased cleavageof dsRNA substrate can be performed in accordance with the teachings ofthe examples of the present application. Agents identified as modulatorsof the level or activity of this enzyme may be useful.

Antisense modulators of human RNase III are provided herein and may beused diagnostically, therapeutically and for research purposes.

The following nonlimiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1 cDNA Cloning

An internet search of the XREF database in the National Center ofBiotechnology Information (NCBI) yielded a 393 base pair (bp) humanexpressed sequenced tag (EST, GenBank accession AA083888), homologous tothe yeast RNase III (RNTI) gene (GenBank accession #AAB04172; SEQ ID NO:5) and the C. elegans RNase III gene (GenBank accession 001326; SEQ IDNO: 3). Three sets of oligonucleotide primers encoding the human RNase HEST sequence were synthesized. Sequence-specific primer sets listed inTable 2 were designed based on the human expressed tag sequence or earlycloned cDNA fragments. These are shown in Table 2. These primers wereused in polymerase chain reaction for 3′ and 5′ RACE and/or fordetection on Southern blots.

TABLE 2 RNase III Oligonucleotide Primers Primer Sequence Position infull SEQ ID Primer name source length cDNA NO Sequence NIII-2 ESTAA083888 3516-3550 21 CCAAATACTGATCGACAACTTATTGAAACTT CTCC NIII-4 ESTAA083888 3569-3606 22 GAGTTTGAAGAAGCAATTGGAGTAATTTTT ACTCATG NIII-6 ESTAA083888 3607-3634 23 TCGACTTCTGGCAAGGGCATTCACATT 3RACE3 Clone #3-42708-2683 24 CCTCTGTGCCAGCTTCTGTTTGTCAG 3RACE2 Clone #3-4 2688-2663 25TGTCAGTTTGTTTGACTTTGGGACTA 3RACE1 Clone #3-4 2662-2637 26TTTGCTAGGAGGTGGCGAAGTTTCAC RACE4 Clone #L40 1923-1894 27GCTTGATGGCCTCTTCTCCAGGATAAATGC RACE5 Clone #L40 1898-1869 28AATGCTGTGCCTAATTCCTGTGCGTCTTGC RACE Det Clone #L40 1723-1676 29CAGGTGCTGTCCTCATCAGACTCACACTCGG ATTCACTGGAACTCTCT 33G Clone #25 831-80630 CACTGGGCAGGAAAGAACTAGGGTTG 33H Clone #25 802-776 31TGGAAACTATTAAAACTGGGAGGTGG 33 Det Clone #25 701-652 32AGGCATGGAGGGAGGGGGCATCATGAAGG GGAAAGTGCCTTGTCCAGGAGBy 3′ RACE (rapid amplification of 3′cDNA), the human RNase III cDNA 3′from the expressed tag sequence was amplified by PCR using humanMarathon ready cDNA (Clontech, Palo Alto Calif.) as templates, andNIII-2/AP1 (for the first amplification) and NIII-4/AP2 (for the secondamplification) as primers. AP1 and AP2 are primers provided with theMarathon ready cDNA by the manufacturer. The standard DNA polymerasechain reaction (PCR) procedure was performed using native pfu DNApolymerase (Stratagene, San Diego Calif.) and its reaction buffer. Theannealing temperature was 55-60° C. The elongation time wasapproximately 6-8 min. The fragments were subjected to agarose gelelectrophoresis. The fragments were subjected to agarose gelelectrophoresis in the TAE buffer, denatured in 0.5 M NaOH and thenelectronically transferred to a nitrocellulose membrane (Bio-Rad,Hercules, Calif.) for confirmation by Southern blot. Southern blots wereperformed using [³²P]-end labeled NIII-6 oligonucleotide as a probe inhybridization buffer (6×SSC, 5×Denhardts solution) containing 100 μg/mlsheared denatured salmon sperm DNA, 0.5% SDS, 10 mM EDTA at 46° C. for 4hr, then washed twice with 1×SSC and 0.1% SDS at 42-59° C. for 20 min.The confirmed fragments were excised from the agarose gel and purifiedby gel extraction (Qiagen, Germany), then subcloned into a zero-bluntvector (Invitrogen, Carlsbad, Calif.) and subjected to DNA sequencing.

Example 2 Screening of the cDNA Libraries, DNA Sequencing and SequenceAnalysis

A human liver cDNA lambda phage Uni-ZAP library (Stratagene, La Jolla,Calif.) was screened using the RACE products as specific probes. Severalpositive clones were isolated. The two longest clones, 3-1 and 3-4,correspond to the COOH-terminal region, nucleotides 2636-3912 and3350-4764, respectively, of the full length cDNA. With primers (3RACE1,3RACE2 and 3RACE3) based on the NH₂-terminal portion of the clone 3-4,5′RACE was performed to clone a cDNA (clone L40) of approximately 1 kb,which encodes the middle part (nucleotides 1661-2688) of the full lengthcDNA. In the same way, a cDNA (clone 25) of the NH₂-terminal portion(nucleotides 645-1898) was cloned. Using clone 25 to screen the liverlibrary again, several clones were isolated, but none includedadditional NH₂-terminal sequence. The most NH₂-terminal clone (328)corresponded to nucleotides 799-2191. The last 5′ RACE was performedwith primers 33G, 33H and 33Dec, based on clone 25, and the NH₂-terminalportion of the cDNA (clone 81, corresponding to nucleotides 1-802) wasgenerated.

The positive cDNA clones were excised into pBluescript phagemid fromlambda phage and subjected to DNA sequencing. Sequencing of the positiveclones was performed with an automatic DNA sequencer by Retrogen Inc.(San Diego, Calif.). The overlapping sequences were aligned and combinedby the assembling program of MacDNASISv3.0 (Hitachi Software EngineeringCo., America, Ltd.) to give the full length (4764 nucleotides)polynucleotide sequence (SEQ ID NO: 1). Protein structure and analysiswere performed by the program MacVector v6.0 (Oxford Molecular Group,UK). A homology search was performed on the NCBI database.

Example 3 Antisense Treatment

HeLa cells were transfected with oligonucleotide mixed with Lipofectin(GIBCO BRL, Gaithersburg, Md.) at a concentration of 37.5-300 nM for 5hours in Opti-MEM (GIBCO BRL). After removing the medium containingoligonucleotide, cells were cultured in DMEM for times indicated andharvested for analysis. Inhibition by antisense oligonucleotides isexpressed compared to control (without oligonucleotide treatment).

Example 4 Northern Hybridization

Total RNA was isolated from HeLa cells using the guanidineisothiocyanate method (R. E. Kingston, in Current protocols in molecularbiology, F. M. Ausubel, et al., Eds., John Wiley & Sons Inc., New York,1997, vol. 1, pp. 4.2.3-4.2.5.). Fifteen μg of total RNA was separatedon a 1% agarose/formaldehyde gel and transferred to Hybond-N+ (Amersham,Arlington Heights, Ill.) followed by fixing using UV crosslinker(Stratagene, La Jolla, Calif.). To detect RNase III mRNA, hybridizationwas performed by using ³²P-labeled human RNase III cDNA in Quik-Hybbuffer (Stratagene, La Jolla, Calif.) at 68° C. for 2 hours. Afterhybridization, membranes were washed in a final stringency of0.1×SSC/0.1% SDS at 60° C. for 30 minutes. Membranes were analyzed usinga PhosphorImager Storm 860 (Molecular Dynamics, Sunnyvale, Calif.). Thelevel of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was usedto normalize the amount of total RNA loaded.

For Northern hybridization of pre-rRNAs, HeLa cells were treated withISIS 25691 and ISIS 27110 for 24 hours using ³²P-end labeled oligoprobes 5′ETS-1 (5′-CAA GGC ACG CCT CTC AGA TCG CTA GAG AAG GCT TTT CTCA-3′; SEQ ID NO: 33), corresponding to 5′ETS and 5.8S-1(5′-CAT TAA TTCTCG CAG CTA GCG CTG CGT TCT TCA TCG ACG C-3′; SEQ ID NO: 34),corresponding to 5.8S rRNA. Hybridizations were performed at 40° C. for2 hours and washed in 2×SSC/0.1% SDS at 40° C. for 1 hour. All otherswere as described above. Data were mean±SD of triplicate determinationof representative experiment.

Example 5 Western Blot Analysis of Human RNase III

Nuclear and non-nuclear fractions from HeLa cells were prepared asdescribed (Dignam et al., Nucleic Acids Res 1983, 11, 1475-89. Wholecell, non-nuclear and nuclear fractions were boiled in SDS-samplebuffer. Then the samples were separated by SDS-PAGE using 4-20%Tris-glycine gels (NOVEX, San Diego, Calif.) under reducing conditions.Molecular weight prestained markers were used (NOVEX) to determine theprotein sizes. The proteins were electrophoretically transferred to aPVDF-membrane and processed for immunoblotting using affinity purifiedanti-SR peptide antibody at 5 μg/ml. The immunoreactive bands werevisualized using the enhanced chemiluminescence method (Amersham,Arlington Heights, Ill.) and analyzed using a PhosphorImager Storm 860(Molecular Dynamics, Sunnyvale, Calif.).

Example 6 Antibody Production

Antibodies were prepared to peptides synthesized having amino acidsequences contained within the SR domain and the III domain of humanRNase III. The SR domain peptide (H—CRSDYDRGRTPSRHRSYERS-OH, amino acids226 to 284; SEQ ID NO: 35) and the III region peptide(H—CRWEREHQEREPDETEDIKK-OH, amino acids 1356 to 1374; SEQ ID NO: 36)were synthesized, coupled to diphtheria toxoid throughmaleimidocaproyl-N-hydroxysuccinamide (MCS), mixed with Freund'sadjuvant (complete for first immunization, incomplete for remainingimmunizations) and injected intramuscularly into New Zealand Whiterabbits. Serum was collected after the second immunization. Antibodytiter was measured by ELISA. Anti-SR and anti-III peptide IgGs wereaffinity purified with SR and III peptides coupled tothiopropyl-Sepharose 6B, respectively.

Example 7 Indirect Immunofluorescence Staining of Human RNase III

HeLa cells were cultured in chamber slides for immunostaining. Cellswere washed once with Dulbecco's Phosphate Buffered Saline (D-PBS,pH7.0), and then fixed in 10% neutral-buffered formalin for 10 minutesfollowed by washing three times with D-PBS. Fixed cells were thenblocked for 30 minutes with 20% fetal bovine serum plus 0.5% Tween 20.Cells were first stained with anti-III peptide antibody (10 μg/ml) for 1hour at 37° C., washed three times with D-PBS plus 0.1% NP-40, andincubated for 1 hour at 37° C. with the FITC goat anti-rabbit IgG(Jackson ImmunoResearch Laboratory, Inc. West Grove, Pa.). The cellswere washed with D-PBS three times and mounted in mounting medium(Vector, Burlingame, Calif.) for examination under a fluorescencemicroscope. NR IgG: normal rabbit IgG was used as control.

Example 8 Indirect Immunofluorescence Staining of Human RNase III inHeLa Cells in Different Phases of the Cell Cycle

HeLa cells were synchronized at early-S phase using the double thymidinemethod (Johnson et al., in The Cell Cycle: A Practical Approach P.Fantes, R. Brooks, Eds., IRL Press, 1993, pp. 1-24). Briefly, cells werecultured in Dulbecco's Modified Eagle Medium (DMEM, 10% fetal calfserum) containing 2 mM of thymidine for 17 hours. After washing twicewith D-PBS, cells were cultured in DMEM for 9 hours followed by secondthymidine treatment for 15 hours. Synchronized cells were then washedtwice with D-PBS, cultured and harvested at 0, 2, 4, 6, 8 and 24 hoursfor immunofluorescence staining and FACS analysis. HeLa cells weredetached from culture flasks with trypsin-EDTA and washed once withD-PBS containing 5 mM of EDTA. Cells were then fixed in 70% ethanol for1 to 24 hours at 4° C. followed by propidium iodine (PI, 50 μg/ml)staining for 1 hour at room temperature. Cell counts (Y axis) and PIcontent (X axis) were determined by FACS analysis (Becton Dickinson andCo., San Jose, Calif.).

Example 9 Expression of GST-RNase III Domain Fusion Protein

A cDNA fragment encoding the human RNase III-like domain(C-terminal-most 466 amino acids) was amplified by PCR and introducedinto a BamH I site upstream and Not I site downstream. This fragment wasfurther subcloned into the sites of the expression vector pGEX-4T-1(Pharmacia Biotech, Piscataway, N.J.) to produce the RNase III fusionprotein with Glutathione S-transferase (GST) at its N-terminus. Theidentity of the construct was proven by DNA sequencing. The GST-RNaseIII fusion protein was expressed in E. coli strain BL21 and purifiedusing glutathione agarose (Pharmacia Biotech, Piscataway, N.J.) undernative conditions with B-PER bacterial protein extraction reagent(Pierce, Rockford, Ill.). Control GST protein was also prepared inparallel from the pGEX-4T-1 plasmid. The purified products wereidentified by Coomassie staining after 12% SDS-polyacrylamide gelelectrophoresis and Western blot analyses with anti-RNase III peptideantibody (see examples above).

Example 10 In Vitro Cleavage of dsRNA

The dsRNA substrate was generated by hybridization of two complementarystrands of RNA produced with T7 and T3 polymerase transcription of thepolylinker region of the pBluscript II KS(−) plasmid (Stratagen, SanDiego, Calif.). The plasmid was digested with either Sst I or Kpn I andfurther purified with phenol/chloroform extraction and ethanolprecipitation. The Sst I or Kpn I-digested plasmids were thentranscribed using T7 or T3 RNA polymerase respectively (Stratagene, SanDiego, Calif.) with or without ³²P-αUTP. The resulting transcribed RNAs(about 100 nt) were purified by electrophoresis on 6% denaturingpolyacrylamide gel. The ³²P radiolabeled T7 transcript and unlabeled T3transcript fragments were mixed and heated for 5 min at 90° C. in abuffer containing 20 mM KCl, 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA. MgCl,BSA and RNase inhibitor were added to the mixture after heating (finalconcentrations were 10 mM. 100 ng/ml and 10 unit/ml respectively). Themixture was incubated at 37° C. for 2 hr and the duplex RNA was purifiedon 6% non-denaturing gels. The ³²P-labelled T7 transcript was also usedas the ssRNA control substrate. To evaluate cleavage, 0.4 μg of GSTprotein or GST-RNase III (approximately 5-10 pmole of purified GST-RNaseIII) fusion protein was incubated with labeled dsRNA (250,000 cpm)(approximately 5-10 fmole) and ssRNA (250,000 cpm) at 37° C. in a buffercontaining 20 mM KCl, 50 mM Tris-HCl (pH 7.5), 5 mM MgCl, 50 mM NaCl,0.1 mM DTT, 0.1 mg/ml yeast tRNA and 10 unit/ml RNase inhibitor in thetotal volume of 60 μl. The digested samples were quenched at specifictimes and analyzed using non-denaturing polyacrylamide gelelectrophoresis and PhosphorImager analysis.

1-64. (canceled)
 65. A method comprising contacting a cell with amodified oligonucleotide wherein: the modified oligonucleotide is 12 to30 nucleobases in length; the modified oligonucleotide is complementaryto nucleobases 3051 to 4004 of SEQ ID NO: 1 or to nucleobases 4197 to4397 of SEQ ID NO:
 1. 66. The method of claim 65 wherein the modifiedoligonucleotide is complementary to nucleobases 3051 to 4004 of SEQ IDNO:
 1. 67. The method of claim 66 wherein in the modifiedoligonucleotide comprises the nucleobase sequence of SEQ ID NO: 8 or 9.68. The method of claim 65 wherein the modified oligonucleotide iscomplementary to nucleobases 4197 to 4397 of SEQ ID NO:
 1. 69. Themethod of claim 68 wherein the modified oligonucleotide comprises thenucleobase sequence of SEQ ID NO: 12, 13, 14, or
 15. 70. The method ofclaim 65, wherein the modified oligonucleotide comprises at least onenucleoside comprising a modified sugar.
 71. The method of claim 70,wherein at least one modified nucleoside comprises a 2′-O-methoxyethyl.72. The method of claim 65 wherein the modified oligonucleotidecomprises at least one modified internucleoside linkage.
 73. The methodof claim 65, wherein the modified oligonucleotide comprises: a gapsegment consisting of linked deoxynucleosides; a 5′ wing segmentconsisting of linked modified nucleosides; and a 3′ wing segmentconsisting of linked modified nucleosides; wherein the gap segment ispositioned between the 5′ wing segment and the 3′ wing segment andwherein each modified nucleoside of each wing segment comprises amodified sugar.
 74. The method of claim 73, wherein each modified sugarcomprises a 2′-modification.
 75. The method of claim 74, wherein each2′-modification is a 2′-O-methoxyethyl modification.
 76. The method ofclaim 65, wherein the cell is in an animal.
 77. The method of claim 76,wherein the animal is a mammal.
 78. The method of claim 77, wherein theanimal is a human.